Nanopore-based sequencing with varying voltage stimulus

ABSTRACT

A method of analyzing a molecule in a nanopore is disclosed. A voltage is applied across a nanopore that is inserted in a membrane by coupling the nanopore to a voltage source. The nanopore is decoupled from the voltage source. After the decoupling, a rate of decay of the voltage across the nanopore is determined. A molecule in the nanopore is distinguished from other possible molecules based on the determined rate of decay of the voltage across the nanopore.

CROSS REFERENCE TO OTHER APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/371,106, now U.S. Pat. No. 10,215,731, entitled NANOPORE-BASEDSEQUENCING WITH VARYING VOLTAGE STIMULUS, filed Dec. 6, 2016, which is acontinuation of U.S. patent application Ser. No. 14/577,511, now U.S.Pat. No. 9,557,294, entitled NANOPORE-BASED SEQUENCING WITH VARYINGVOLTAGE STIMULUS, filed Dec. 19, 2016, both of which are incorporatedherein by reference for all purposes.

BACKGROUND OF THE INVENTION

Advances in micro-miniaturization within the semiconductor industry inrecent years have enabled biotechnologists to begin packingtraditionally bulky sensing tools into smaller and smaller form factors,onto so-called biochips. It would be desirable to develop techniques forbiochips that make them more robust, efficient, and cost-effective.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip.

FIG. 2 illustrates an embodiment of a cell 200 performing nucleotidesequencing with the Nano-SBS technique.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags.

FIG. 5 illustrates an embodiment of a circuitry 500 in a cell of ananopore based sequencing chip.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state.

FIG. 7A illustrates an additional embodiment of a circuitry 700 in acell of a nanopore based sequencing chip, wherein the voltage appliedacross the nanopore can be configured to vary over a time period duringwhich the nanopore is in a particular detectable state.

FIG. 7B illustrates an additional embodiment of a circuitry 701 in acell of a nanopore based sequencing chip, wherein the voltage appliedacross the nanopore can be configured to vary over a time period duringwhich the nanopore is in a particular detectable state.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane.

FIG. 9 illustrates an embodiment of a plot of the voltage applied acrossthe nanopore versus time when process 800 is performed and repeatedthree times.

FIG. 10 illustrates an embodiment of the plots of the voltage appliedacross the nanopore versus time when the nanopore is in differentstates.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims andthe invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

Nanopore membrane devices having pore sizes on the order of onenanometer in internal diameter have shown promise in rapid nucleotidesequencing. When a voltage potential is applied across a nanoporeimmersed in a conducting fluid, a small ion current attributed to theconduction of ions across the nanopore can be observed. The size of thecurrent is sensitive to the pore size.

A nanopore based sequencing chip may be used for DNA sequencing. Ananopore based sequencing chip incorporates a large number of sensorcells configured as an array. For example, an array of one million cellsmay include 1000 rows by 1000 columns of cells.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip. A membrane 102 is formed over the surface of the cell.In some embodiments, membrane 102 is a lipid bilayer. The bulkelectrolyte 114 containing soluble protein nanopore transmembranemolecular complexes (PNTMC) and the analyte of interest is placeddirectly onto the surface of the cell. A single PNTMC 104 is insertedinto membrane 102 by electroporation. The individual membranes in thearray are neither chemically nor electrically connected to each other.Thus, each cell in the array is an independent sequencing machine,producing data unique to the single polymer molecule associated with thePNTMC. PNTMC 104 operates on the analytes and modulates the ioniccurrent through the otherwise impermeable bilayer.

With continued reference to FIG. 1, analog measurement circuitry 112 isconnected to a metal electrode 110 covered by a thin film of electrolyte108. The thin film of electrolyte 108 is isolated from the bulkelectrolyte 114 by the ion-impermeable membrane 102. PNTMC 104 crossesmembrane 102 and provides the only path for ionic current to flow fromthe bulk liquid to working electrode 110. The cell also includes acounter electrode (CE) 116, which is an electrochemical potentialsensor. The cell also includes a reference electrode 117.

In some embodiments, a nanopore array enables parallel sequencing usingthe single molecule nanopore-based sequencing by synthesis (Nano-SBS)technique. FIG. 2 illustrates an embodiment of a cell 200 performingnucleotide sequencing with the Nano-SBS technique. In the Nano-SBStechnique, a template 202 to be sequenced and a primer are introduced tocell 200. To this template-primer complex, four differently taggednucleotides 208 are added to the bulk aqueous phase. As the correctlytagged nucleotide is complexed with the polymerase 204, the tail of thetag is positioned in the barrel of nanopore 206. The tag held in thebarrel of nanopore 206 generates a unique ionic blockade signal 210,thereby electronically identifying the added base due to the tags'distinct chemical structures.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags. A nanopore 301 is formed in a membrane302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) isassociated with the nanopore. In some cases, polymerase 303 iscovalently attached to nanopore 301. Polymerase 303 is associated with anucleic acid molecule 304 to be sequenced. In some embodiments, thenucleic acid molecule 304 is circular. In some cases, nucleic acidmolecule 304 is linear. In some embodiments, a nucleic acid primer 305is hybridized to a portion of nucleic acid molecule 304. Polymerase 303catalyzes the incorporation of nucleotides 306 onto primer 305 usingsingle stranded nucleic acid molecule 304 as a template. Nucleotides 306comprise tag species (“tags”) 307.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags. Stage A illustrates the components asdescribed in FIG. 3. Stage C shows the tag loaded into the nanopore. A“loaded” tag may be one that is positioned in and/or remains in or nearthe nanopore for an appreciable amount of time, e.g., 0.1 millisecond(ms) to 10000 ms. In some cases, a tag that is pre-loaded is loaded inthe nanopore prior to being released from the nucleotide. In someinstances, a tag is pre-loaded if the probability of the tag passingthrough (and/or being detected by) the nanopore after being releasedupon a nucleotide incorporation event is suitably high, e.g., 90% to99%.

At stage A, a tagged nucleotide (one of four different types: A, T, G,or C) is not associated with the polymerase. At stage B, a taggednucleotide is associated with the polymerase. At stage C, the polymeraseis docked to the nanopore. The tag is pulled into the nanopore duringdocking by an electrical force, such as a force generated in thepresence of an electric field generated by a voltage applied across themembrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with thenucleic acid molecule. These non-paired nucleotides typically arerejected by the polymerase within a time scale that is shorter than thetime scale for which correctly paired nucleotides remain associated withthe polymerase. Since the non-paired nucleotides are only transientlyassociated with the polymerase, process 400 as shown in FIG. 4 typicallydoes not proceed beyond stage D. For example, a non-paired nucleotide isrejected by the polymerase at stage B or shortly after the processenters stage C.

Before the polymerase is docked to the nanopore, the conductance of thenanopore is ˜300 pico Siemens (300 pS). At stage C, the conductance ofthe nanopore is about 60 pS, 80 pS, 100 pS, or 120 pS corresponding toone of the four types of tagged nucleotides. The polymerase undergoes anisomerization and a transphosphorylation reaction to incorporate thenucleotide into the growing nucleic acid molecule and release the tagmolecule. In particular, as the tag is held in the nanopore, a uniqueconductance signal (e.g., see signal 210 in FIG. 2) is generated due tothe tag's distinct chemical structures, thereby identifying the addedbase electronically. Repeating the cycle (i.e., stage A through E orstage A through F) allows for the sequencing of the nucleic acidmolecule. At stage D, the released tag passes through the nanopore.

In some cases, tagged nucleotides that are not incorporated into thegrowing nucleic acid molecule will also pass through the nanopore, asseen in stage F of FIG. 4. The unincorporated nucleotide can be detectedby the nanopore in some instances, but the method provides a means fordistinguishing between an incorporated nucleotide and an unincorporatednucleotide based at least in part on the time for which the nucleotideis detected in the nanopore. Tags bound to unincorporated nucleotidespass through the nanopore quickly and are detected for a short period oftime (e.g., less than 10 ms), while tags bound to incorporatednucleotides are loaded into the nanopore and detected for a long periodof time (e.g., at least 10 ms).

FIG. 5 illustrates an embodiment of a circuitry 500 in a cell of ananopore based sequencing chip. As mentioned above, when the tag is heldin nanopore 502, a unique conductance signal (e.g., see signal 210 inFIG. 2) is generated due to the tag's distinct chemical structures,thereby identifying the added base electronically. The circuitry in FIG.5 maintains a constant voltage across nanopore 502 when the current flowis measured. In particular, the circuitry includes an operationalamplifier 504 and a pass device 506 that maintain a constant voltageequal to V_(a) or V_(b) across nanopore 502. The current flowing throughnanopore 502 is integrated at a capacitor n_(cap) 508 and measured by anAnalog-to-Digital (ADC) converter 510.

However, circuitry 500 has a number of drawbacks. One of the drawbacksis that circuitry 500 only measures unidirectional current flow. Anotherdrawback is that operational amplifier 504 in circuitry 500 mayintroduce a number of performance issues. For example, the offsetvoltage and the temperature drift of operational amplifier 504 may causethe actual voltage applied across nanopore 502 to vary across differentcells. The actual voltage applied across nanopore 502 may drift by tensof millivolts above or below the desired value, thereby causingsignificant measurement inaccuracies. In addition, the operationalamplifier noise may cause additional detection errors. Another drawbackis that the portions of the circuitry for maintaining a constant voltageacross the nanopore while current flow measurements are made arearea-intensive. For example, operational amplifier 504 occupiessignificantly more space in a cell than other components. As thenanopore based sequencing chip is scaled to include more and more cells,the area occupied by the operational amplifiers may increase to anunattainable size. Unfortunately, shrinking the operational amplifier'ssize in a nanopore based sequencing chip with a large-sized array mayraise other performance issues. For example, it may exacerbate theoffset and noise problems in the cells even further.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state. One of the possible statesof the nanopore is an open-channel state when a tag-attachedpolyphosphate is absent from the barrel of the nanopore. Another fourpossible states of the nanopore correspond to the states when the fourdifferent types of tag-attached polyphosphate (A, T, G, or C) are heldin the barrel of the nanopore. Yet another possible state of thenanopore is when the membrane is ruptured. FIGS. 7A and 7B illustrateadditional embodiments of a circuitry (700 and 701) in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state. In the above circuits, theoperational amplifier is no longer required.

FIG. 6 shows a nanopore 602 that is inserted into a membrane 612, andnanopore 602 and membrane 612 are situated between a cell workingelectrode 614 and a counter electrode 616, such that a voltage isapplied across nanopore 602. Nanopore 602 is also in contact with a bulkliquid/electrolyte 618. Note that nanopore 602 and membrane 612 aredrawn upside down as compared to the nanopore and membrane in FIG. 1.Hereinafter, a cell is meant to include at least a membrane, a nanopore,a working cell electrode, and the associated circuitry. In someembodiments, the counter electrode is shared between a plurality ofcells, and is therefore also referred to as a common electrode. Thecommon electrode can be configured to apply a common potential to thebulk liquid in contact with the nanopores in the measurements cells. Thecommon potential and the common electrode are common to all of themeasurement cells. There is a working cell electrode within eachmeasurement cell; in contrast to the common electrode, working cellelectrode 614 is configurable to apply a distinct potential that isindependent from the working cell electrodes in other measurement cells.

In FIGS. 7A and 7B, instead of showing a nanopore inserted in a membraneand the liquid surrounding the nanopore, an electrical model 702representing the electrical properties of the nanopore and the membraneis shown. Electrical model 702 includes a capacitor 706 that models acapacitance associated with the membrane and a resistor 704 that modelsa resistance associated with the nanopore in different states (e.g., theopen-channel state or the states corresponding to having different typesof tag/molecule inside the nanopore). Note in FIGS. 7A and 7B that therespective circuitry does not require an extra capacitor (e.g., n_(cap)508 in FIG. 5) that is fabricated on-chip, thereby facilitating thereduction in size of the nanopore based sequencing chip.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane. Process 800 may be performed using the circuitries shown inFIG. 6, 7A, or 7B. FIG. 9 illustrates an embodiment of a plot of thevoltage applied across the nanopore versus time when process 800 isperformed and repeated three times. As will be described in greaterdetail below, the voltage applied across the nanopore is not heldconstant. In contrast, the voltage applied across the nanopore changesover time. The rate of the voltage decay (i.e., the steepness of theslope of the applied voltage across the nanopore versus time plot)depends on the cell resistance (e.g., the resistance of resistor 704 inFIG. 7A). More particularly, as the resistance associated with thenanopore in different states (e.g., the open-channel state, the statescorresponding to having different types of tag/molecule inside thenanopore, and the state when the membrane is ruptured) are different dueto the molecules'/tags' distinct chemical structure, differentcorresponding rates of voltage decay may be observed and thus may beused to identify the different states of the nanopore.

With reference to FIG. 8 and FIG. 7A, at 802 of process 800, a voltageis applied across the nanopore by coupling the nanopore to a voltagesource. For example, as shown in FIG. 7A, a voltage V_(pre) 710 isapplied to the cell working electrode when a switch S1 708 is closed. Asshown in FIG. 9, the initial voltage applied across the nanopore isV_(pre)-V_(liquid), where V_(liquid) is the voltage of the bulk liquidin contact with the nanopore. As the voltage source is connected to theworking electrode, the capacitor associated with the membrane is chargedand energy is stored in an electric field across the membrane.

At 804 of process 800, the capacitor associated with the membrane(capacitor 706) is discharged by decoupling the nanopore and themembrane from the voltage source, and the energy stored in the electricfield across the membrane is thereby dissipated. For example, as shownin FIG. 7A, the voltage source is disconnected when switch S1 708 isopened. After switch S1 708 is opened, the voltage across the nanoporebegins to decay exponentially, as shown in FIG. 9. The exponential decayhas a RC time constant τ=RC, where R is the resistance associated withthe nanopore (resistor 704) and C is the capacitance associated with themembrane (capacitor 706) in parallel with R.

At 806 of process 800, a rate of the decay of the voltage applied acrossthe nanopore is determined. The rate of the voltage decay is thesteepness of the slope of the applied voltage across the nanopore versustime curve, as shown in FIG. 9. The rate of the voltage decay may bedetermined in different ways.

In some embodiments, the rate of the voltage decay is determined bymeasuring a voltage decay that occurs during a fixed time interval. Forexample, as shown in FIG. 9, the voltage applied at the workingelectrode is first measured by ADC 712 at time t₁, and then the voltageis again measured by ADC 712 at time t₂. The voltage differenceΔV_(applied) is greater when the slope of the voltage across thenanopore versus time curve is steeper, and the voltage differenceΔV_(applied) is smaller when the slope of the voltage curve is lesssteep. Thus, ΔV_(applied) may be used as a metric for determining therate of the decay of the voltage applied across the nanopore. In someembodiments, to increase the accuracy of the measurement of the rate ofvoltage decay, the voltage may be measured additional times at fixedintervals. For example, the voltage may be measured at t₃, t₄, and soon, and the multiple measurements of ΔV_(apphed) during the multipletime intervals may be jointly used as a metric for determining the rateof the decay of the voltage applied across the nanopore. In someembodiments, to increase the accuracy of the measurement of the rate ofvoltage decay, correlated double sampling (CDS) may be used.

In some embodiments, the rate of the voltage decay is determined bymeasuring a time duration that is required for a selected amount ofvoltage decay. In some embodiments, the time required for the voltage todrop from a fixed voltage V₁ to a second fixed voltage V₂ may bemeasured. The time required is less when the slope of the voltage curveis steeper, and the time required is greater when the slope of thevoltage curve is less steep. Thus, the measured time required may beused as a metric for determining the rate of the decay of the voltageapplied across the nanopore.

At 808 of process 800, a state of the nanopore is determined based onthe determined rate of voltage decay. One of the possible states of thenanopore is an open-channel state during which a tag-attachedpolyphosphate is absent from the barrel of the nanopore. Other possiblestates of the nanopore correspond to the states when different types ofmolecules are held in the barrel of the nanopore. For example, anotherfour possible states of the nanopore correspond to the states when thefour different types of tag-attached polyphosphate (A, T, G, or C) areheld in the barrel of the nanopore. Yet another possible state of thenanopore is when the membrane is ruptured. The state of the nanopore canbe determined based on the determined rate of voltage decay, because therate of the voltage decay depends on the cell resistance; i.e., theresistance of resistor 704 in FIG. 7A. More particularly, as theresistance associated with the nanopore in different states aredifferent due to the molecules/tags' distinct chemical structure,different corresponding rates of voltage decay may be observed and thusmay be used to identify the different states of the nanopore.

FIG. 10 illustrates an embodiment of the plots of the voltage appliedacross the nanopore versus time when the nanopore is in differentstates. Curve 1002 shows the rate of voltage decay during anopen-channel state. In some embodiments, the resistance associated withthe nanopore in an open-channel state is in the range of 100 Mohm to 20Gohm. Curves 1004, 1006, 1008, and 1010 show the different rates ofvoltage decay corresponding to the four capture states when the fourdifferent types of tag-attached polyphosphate (A, T, G, or C) are heldin the barrel of the nanopore. In some embodiments, the resistanceassociated with the nanopore in a capture state is within the range of200 Mohm to 40 Gohm. Note that the slope of each of the plots isdistinguishable from each other.

At 810 of process 800, it is determined whether process 800 is repeated.For example, the process may be repeated a plurality of times to detecteach state of the nanopore. If the process is not repeated, then process800 terminates; otherwise, the process restarts at 802 again. At 802, avoltage is reasserted across the nanopore by connecting to the voltagesource. For example, as shown in FIG. 7A, a voltage V_(pre) 710 isapplied across the nanopore when switch S1 708 is closed. As shown inFIG. 9, the applied voltage jumps back up to the level of V_(pre). Asprocess 800 is repeated a plurality of times, a saw-tooth like voltagewaveform is applied across the nanopore over time. FIG. 9 alsoillustrates an extrapolation curve 904 showing the RC voltage decay overtime had the voltage V_(pre) 710 not been reasserted.

As shown above, configuring the voltage applied across the nanopore tovary over a time period during which the nanopore is in a particulardetectable state has many advantages. One of the advantages is that theelimination of the operational amplifier, the pass device, and thecapacitor (e.g., n_(cap) 508 in FIG. 5) that are otherwise fabricatedon-chip in the cell circuitry significantly reduces the footprint of asingle cell in the nanopore based sequencing chip, thereby facilitatingthe scaling of the nanopore based sequencing chip to include more andmore cells (e.g., having millions of cells in a nanopore basedsequencing chip). The capacitance in parallel with the nanopore includestwo portions: the capacitance associated with the membrane and thecapacitance associated with the integrated chip (IC). Due to the thinnature of the membrane, the capacitance associated with the membranealone can suffice to create the required RC time constant without theneed for additional on-chip capacitance, thereby allowing significantreduction in cell size and chip size.

Another advantage is that the circuitry of a cell does not suffer fromoffset inaccuracies because V_(pre) is applied directly to the workingelectrode without any intervening circuitry. Another advantage is thatsince no switches are being opened or closed during the measurementintervals, the amount of charge injection is minimized.

Furthermore, the technique described above operates equally well usingpositive voltages or negative voltages. Bidirectional measurements havebeen shown to be helpful in characterizing a molecular complex. Inaddition, bidirectional measurements are required when the type of ionicflow that is driven through the nanopore is via non-faradaic conduction.Two types of ionic flow can be driven through the nanopore-faradaicconduction and non-faradaic conduction. In faradaic conduction, achemical reaction occurs at the surface of the metal electrode. Thefaradaic current is the current generated by the reduction or oxidationof some chemical substances at an electrode. The advantage ofnon-faradaic conduction is that no chemical reaction happens at thesurface of the metal electrode.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

What is claimed is:
 1. A method of analyzing a molecule in a nanopore,the method comprising: applying a voltage between a working electrodeand a counter electrode, wherein a membrane having a protein nanoporedisposed therein is located between the working electrode and thecounter electrode, wherein the applied voltage is configured totranslocate the molecule or a portion of the molecule into or throughthe protein nanopore; measuring a rate of decay in a voltage across theprotein nanopore using the working electrode and counter electrode whilethe molecule or the portion of the molecule is located within theprotein nanopore; and distinguishing the molecule in the proteinnanopore from other possible molecules based at least in part on themeasured rate of decay in the voltage across the protein nanopore,wherein the molecule and other possible molecules are modifiednucleotides with a modification that allows one type nucleotide to bedistinguished from the other types of nucleotide.
 2. The method of claim1, further comprising reducing the voltage between the working electrodeand the counter electrode, and wherein the step of measuring the rate ofdecay in the voltage across the protein nanopore occurs after the stepof reducing the voltage between the working electrode and the counterelectrode.
 3. The method of claim 1, wherein the step of measuring therate of decay in the voltage across the protein nanopore comprisesmeasuring the rate of decay in the voltage across the protein nanoporeover a predetermined duration.
 4. The method of claim 1, wherein thestep of measuring the rate of decay in the voltage across the proteinnanopore comprises measuring an amount of time needed to achieve apredetermined rate of decay in the voltage across the protein nanopore.5. The method of claim 1, wherein the molecule is a tag attached to anucleotide.
 6. The method of claim 1, wherein the step of measuring therate of decay in the voltage across the protein nanopore comprisesmeasuring a rate of decay in voltage across a capacitor that isassociated with the membrane.